Objective To study the resistances of CDl33 + subset purified from gastric cancer cell line to chemotherapy drugs and the mechanism of this resistance regarding to the mRNA expressions of both Bcl-2 and BAX in relation to the relative apoptotic genes.Methods CD133 + subset and CD133-subset were purified from KATOⅢ cell linc by magnetic activated cell sorting.The proliferating ability of these two subsets resistantnt to 5-FU,Cisplatin(DDP,PDD) and Etoposide was checked and compared by CCK-8 test.The apoptotic changes of these two subsets regarding to the expression of mRNA of both Bcl-2 and BAX were also analized by RT-PCR.Results In CD133 + subset,the contant percentage of CD133 + expression rate was 90％ via analysis of flow cytometye.Twelve hours after treatment of5-FU,DDP and VP-16,the cells in both CD133 + subgroup and CD133-subgroup would gradually start to change in apoptotic morphology.The growth inhibiting rate by CCK-8 measurement for 5-FU,DDP and VP-16 groups in CD133 + subgroup was significantly lower than that in CD133-subgroup.The data under different treatment respectively was,5-FU:(30.56 ± 1.99) ％-(88.60 ± 1.95) ％ vs (32.81 ± 2.67) ％-(95.73±2.12)％,P=0.045,cisplatin:(45.89 ±3.64)％-(81.20 ± 1.18)％ vs (50.21 ±3.22)％-(90.46±1.89)％,P=0.043,VP-16:(37.21 ±3.80)％-(78.49 ±3.22)％ vs (35.55 ±3.23)％-(89.32 ±-3.54) ％,P =0.048).After treatment of these three kind of anti-tumour drugs,the expression level of Bcl-2 mR-NA decreased significantly and the expression level of BAX mRNA increased significantly in both CD133 + subgroup and CD133-subgroup.However,these changing ranges of Bcl-2 mRNA and BAX mRNA were more obvious in CD133 + subgroup in comparison with those in CD133-subgroup.Conclusions In some degree,resistent potentiality of CD133 + cells to 5-FU,DDP and VP-16 has been identified,which may probably be due to the up-regulation of the expression of BAX and down-regulation of the expression of Bcl-2.

Objective To detect the expression of c-met (hepatocyte growth factor receptor), e2f-1 (transcription factor) and Ki-67 in tissues of gastric cancer, explore the relationship among them, and investigate their correlationship with clinicopathological characteristics and prognosis. Methods The tissue samples of gastric cancer from 86 patients with radical resection were subjected to immunohistochemical staining, and the expression of c-met, e2f-1 and Ki-67 was detected. The relationship between the clinicopathological characteristics and the expression of c-met, e2f-1 and Ki-67, and that among the expression of c-met, e2f-1 and Ki-67 were explored by univariate and multivariate data analysis. And the relationship between prognosis and expression of c-met, e2f-1 and Ki-67 was analysed by Log rank test. Results c-met, e2f-1 and Ki-67 were widely expressed in tissues of gastric cancer. The higher expression of c-met was associated with higher expression of Ki-67, shorter survival time, upper tumor location, higher lymph node metastasis rate, higher N stage and TNM stage (P<0.05), and the lower expression of e2f-1 was associated with larger tumor diameter, higher TNM stage, deeper invasion, higher lymph node metastasis rate and higher N stage (P<0.05). The multivariate analysis revealed that depth of invasion, N stage, TNM stage and expression of Ki-67 were independent factors for positive expression of c-met, and age and survival time were independent factors for positive expression of e2f-1. Log rank test demonstrated that the factors related to survival included expression of c-met, e2f-1 and Ki-67, N stage, tumor diameter, tumor location, lymph node metastasis rate, depth of invasion and TNM stage. Conclusion c-met is an indicator for malignant behavior and poorer prognosis of gastric cancer. There is a higher expression of e2f-1 in early gastric cancer, while advanced gastric cancer may be associated with a lower expression of e2f-1.

Objective To investigate the expression of CD133 mRNA in tissues of gastric cancer,and explore its relationship with clinicopathological features. Methods The tissues of gastric cancer and normal tissues adjacent to gastric cancer were obtained from 31 patients.The expression of CD133 mRNA was detected by semi-quantitative RT-PCR,and its relationship with clinicopathological features such as sex,age,tumor diameter,infiltration depth,TNM staging,tumor differentiation,lymph node metastasis and Ki-67 proliferation index was analysed. Results The relative gray scale values of CD133 mRNA in tissues of gastric cancer and normal tissues adjacent to gastric cancer were 0.378 3±0.141 1 and 0.038 1 ±0.091 9,respectively(P=0.000).The relative gray scale values of CD133 mRNA in tissues of gastric cancer with tumor diameter>5 cm were significantly higher than those with tumor diameter≤5 cm[(0.439 3±0.148 4)vs(0.334 3±0.121 2)](P=0.041),and those in tissues with lymph node metastasis were significantly higher than those without lymph node metastasis[(0.426 6±0.132 0)vs(0.239 5±0.030 9)](P=0.004).The rate of lymph node metastasis and the number of metastatic lymph nodes were positively related to relative gray scale values of CD133 mRNA(r=0.466,P=0.008;r=0.464,P=0.009).The relative gray scale values of CD133 mRNA in those with low expression of Ki-67 were significantly higher than those with high expression of Ki-67[(0.436 4±0.139 8)vs(0.316 4±0.117 4)](P=0.02),and expression of Ki-67 were negatively related to relative gray scale values of CD133 mRNA(r=-0.461,P=0.009).Conclusion The expression of CD133 mRNA in tissues of gastric cancer was associated with the rate of lymph node metastasis,number of metastatic lymph nodes and expression of Ki-67,which reflect the status of lymph node metastasis and proliferation of gastric cancer.

Objective: To investigate the influence of CD133+expression on patients' survival and resistance of CD133+cells to anti-tumor agents in gastric cancer (GC).
Methods: Influence of CD133 expression on prognosis was analyzed employing sam-ples from patients with GC. GC cell lines were utilized to separate CD133+and CD133?subpopulations by immunomagnetic separation and to analyze the biological features of two subpopulations in vitro and in vivo, especially in resistant to anti-tumor reagents and its apoptotic mechanism.
Results: The lower CD133+group showed a significantly better survival compared with the higher CD133+group. The highest content of CD133+subpopulations for KATO-III cells had stronger proliferative ability than CD133?subpopulations. A single CD133+cell was capable of generating new cell colony and the tumorigenicity rate in nude mice was 100% for CD133+ clonal spheres or for CD133+ cells, but 0% for CD133? cells. Furthermore, the higher expression levels of Oct-4, Sox-2, Musashi-1 and ABCG2 in CD133+ clonal spheres were identified compared with CD133+ cells or CD133? cells. Under the treatment of anti-tumor reagents, CD133+ cells had lower suppression rates compared with CD133? cells while lower level of Bcl-2 and higher level of Bax were found in CD133+cells compared with CD133?cells.
Conclusions: The patients with lower CD133+expression had a better survival. Enriched CD133+ cells in clonal sphere shared the ability to be self-renewable, proliferative, tumorigenic and resistant to anti-tumor agents as probably regulated by Bcl-2 and Bax.

Objective To compare the inhibition effects of three synthesized fragments used in small interfering RNA(siRNA) against CD133 gene in KATO-Ⅲ gastric cancer cells,and to study effects of suppressed CD133 on the proliferating ability of intervened cells.Methods Three fragments of siRNA were designed and synthesized targeted at the mRNA of CD133.Cell fluorescence counting under confocal laser scanning microscope was used to determine the transfection efficiency after transfection with the CD133FITC-siRNA.The knock-down effect of the CD133 gene was detected by RT-PCR and Western blotting.CCK-8 (cell counting kit-8 assay) was performed to measure the variation of the cell proliferative viability after the above-mentioned treatment.Results The transfection efficiency of siRNA was (85 ± 8) ％ in KATO Ⅲ Gastric cacer cell.All these three fragments of CD133 siRNA effectively inhibited the expression of CD133 gene,the inhibition rate being (11 ± 2) ％,(19 ± 2) ％,(24 ± 3) ％respectively.Compared with the control group,the cell proliferation viability was restrained (42 ± 4)％ in CD133siRNA-3 group (P ＜0.05).Conclusions CD133siRNAs were successfully transfected into KATO Ⅲ Gastric cacer cells and repressed the expression of CD133.Meanwhile,the CD133siRNA fragment 3 was screened from three CD133 siRNA,which has the best inhibition effect.The results provide preliminary evidence for the intereference of CD133+ gastric cancer cells subsequently.

Objective To investigate the biological effect of epithelial-to-mesenchymal transition (EMT) under the treatment of transforming growth factor (TGF)-β1 on the human gastric cancer cell line SGC7901 in vitro,and to observe whether the TGF-β1 can generate the tumor initiating cells ability in SGC7901 or not.Methods SGC7901 cells were cultured with TGF-β1.The morphological change was observed.The effect on proliferation of SGC7901 cells was detected by CCK-8.The invasion assay was used to investigate the motility and the invasion ability of SGC7901 cells.Immuofluorescence was used to detect the expression of E-cadherin and N-cadherin.The mRNA and protein's expression levels of EMT-related factors and CD44 were analyzed by RT-PCR and Western blotting respectively.Results TGF-β1 induced morphological alterations from epithelial to mesenchymal cells.The proliferation of SGC7901 cells was inhibited,and the ability of motility and invasion of SGC7901 cells were greatly enhanced after being treated with TGF-β1.RT-PCR and Western blotting showed that the expression of Snail (P ＜ 0.05),N-cadherin (P ＜ 0.05) and CD44 (P ＜ 0.05) were significantly increased while the expression of E-cadherin was decreased (P ＜ 0.05).Conclusions TGF-β1 can generate the EMT.The CD44 expression was up-regulated.TGF-β1 can inhibit the proliferation and promote the motility and invasion ability of SGC7901 cells.

Objective To investigate if TGF-β1 induces epithelial-mesenchymal transition (EMT) and promotes the obtaining of stemness characteristics in gastric cancer cell lines.Methods After KATO-Ⅲ cells were cultured with or without 5 ng/mL TGF-β1,the morphological change was observed and compared under phase-contrast microscopy.At the same time,the effect of TGF-β1 on the proliferation of KATO-Ⅲ cells was detected by CCK-8.On the other hand,the mRNA and protein' s expressions of EMT-related factors,ESC markers and TICs markers were analyzed by RT-PCR and Western blotting methods too.Results TGF-β1 induced morphological alterations from epithelial to mesenchymal cells.The proliferation of KATO-Ⅲ cells was inhibited after treated with TGF-β1 (P ＜ 0.05).After treated with TGF-β1,the relative mRNA expression levels of Snail (0.5219 ±0.0147) and N-cadherin(0.6640 ±0.0124) were higher than that in control group(0.2049 ±0.0214,P =0.004,0.2722 ± 0.0098,P =0.001),the relative protein expression levels of Snail (0.4769 ± 0.0234) and N-cadherin (0.5014 ± 0.0216) were higher than that in control group (0.2534 ± 0.0345,P =0.02,0.2026 ± 0.0268,P =0.009),while the relative E-cadherin mRNA and protein levels in TGF-β1 treated group (0.4701 ± 0.0215,0.1349 ± 0.0258) were lower than that in control group (0.6792 ± 0.0157,P =0.01 ; 0.6055 ± 0.0227,P =0.004),while the relative mRNA expressions of ESC markers such as Sox2,OCT4,Nanog in TGF-β1 treated group (0.594 ± 0.039、0.438 ± 0.033、0.489 ± 0.037) were higher than that in control group (0.143 ± 0.013,P =0.001,0.156 ± 0.025,P =0.001,0.325 ± 0.046,P =0.03),the relative mRNA expression levels of CD44 (0.437 ±0.037) and CD133(0.543 ±0.028) were higher than that in control group (0.247 ±0.024,P =0.000,0.139 ± 0.016,P =0.000),the relative protein expression levels of CD44 (0.429 ± 0.034) and CD133 (0.316 ±0.027) in TGF-β1 treated group were higher than that in control group (0.152 ± 0.014,P =0.000,0.110 ±0.010,P =0.000),cloning sphere-forming capacity was greatly enhanced after treated with TGF-β1 (P ＜ 0.01).Conclusion TGF-β1 can induce EMT in KATO-Ⅲ cells and promote the obtaining of stemness characteristics in gastric cancer cell lines.

Objective To investigate the clinicopathologic characters and prognostic value of epithelial mesenchymal transition (EMT) related proteins Snail and chemokine receptors 4(CXCR4) in gastric cancer.Methods The expressions of Snail and CXCR4 in the gastric tissues of 50 patients with gastric cancer were detected by Immunohistochemistry.The relation between the expressions of Snail and CXCR4 and the clinicopathologic characters were analyzed.The relative relationship between Snail expression and CXCR4 expression were identified by Spearman method.The correlation between the expressions of Snail and CXCR4 with the survival was analyzed by Kaplan-Meier method.Results The expression of Snail and CXCR4 expression were positive in 31 of 50 cases (62％) and 29 of 50 cases (58％) respectively.The expression levels of Snail and CXCR4 in the gastric cancer patients with diameter more than 5 cm,worse tissue differentiation,vascular invasion,lymphatic vessel invasion,lymph nodes metastasis,and T3 + T4 staging were significantly higher than those in the patients with diameter less than 5 cm,without vascular invasion,lymphatic vessel invasion,lymph nodes metastasis,and T1 + T2 staging (P ＜ 0.05).The Expression of Snail and CXCR4 was positively Correlated(r =0.330,P =0.014).The co-expression of Snail and CXCR4 was significantly associated with poorer prognosis and was applied as an independent prognostic factor for gastric cancer (P =0.001).Conclusions The co-expression of Snail and CXCR4 was the independent prognostic factor for gastric cancer.The combining effect of EMT and CXCR4 may promote the progressions and metastasis of gastric cancer.Therefore,it may be of an effective way for the metastasis of gastric cancer to adjust these targets of the Snail and CXCR4.

ObjectiveTo investigate the expression of CXCR4 and CD133mRNA in primary lesion of primary gastric adenocarcinoma and the relation with clinicopathological features,and to explore the correlation of CXCR4 and CD133.MethodsThe primary lesion of primary gastric adenocarcinoma and normal tissues adjacent to gastric cancer were obtained from 50 patients.The diction of CXCR4 and CD133 protein expression was detected by the immunohistochemical staining,and the relative gray scale values of CXCR4 and CD133mRNA by semi-quantitative RT-PCR (Fisher' s exact probability method).Their relationship with clinicopathological features was also investigated ( Spearman relation analysis).ResultsThe positive rates of CXCR4 and CD133 protein in gastric cancers were 76.0％ and 66.0％ respectively,which were significantly higher than that in normal tissues adjacent to gastric cancer ( 16.0％ and 10％ ; P =0.000,P =0.000).The increment of relative gray scale values of CXCR4mRNA was associated with the larger tumor diameter,the later TNM stage and the occurrence of lymphatic metastasis( P ＜ 0.05 ).And the larger diameter of tumor,the later TNM stage were associated with the higher relative gray scale values of CD133mRNA (P ＜0.05).The levels of the relative gray scale values of CXCR4 mRNA and CD133mRNA were positively related(r =0.453,P ＜ 0.01 ).ConclusionsThe higher expression of CXCR4 and CD133mRNA correlateswith tumour diameter,TNM stage and lymphatic vessel invasion. The relative gray scale values of CD133mRNA increase with the increment of the relative gray scale values of CXCR4.

OBJECTIVETo explore the relationship between CD133(+) subsets cells in human gastric cancer (GC) and molecules of drug resistance and their sensitivity to 5-FU.

METHODSThree gastric cancer cell lines therein KATO-III(, SGC7901 and MKN45 were sorted by immunomagnetic beads cell sorting method. Then above cell lines were further divided into un-sorted GC cells, CD133(+) subgroup and CD133(-) subgroup. The expressions of CD133, P-gp, Bax and Bcl-2 were determined by RT-PCR, Western blot and immunoflurescence. Meanwhile, the sensitivity to 5-FU of three subgroups was detected by CCK-8 Kit. The apoptosis induced by 5-FU in three subgroups was determined by Hoechst 33258.

RESULTSExpressions of CD133 in three CD133(+) subgroups were significantly higher than those in un-sorted GC cells and CD133(-) subgroup (all P<0.05). Expressions of P-gp and Bcl-2 in the three GC cell lines were different (all P<0.05). There were significant differences of expressions of P-gp, Bcl-2 and Bax among CD133(+) cells, un-sorted GC cells and CD133(-) cells (all P<0.05). CCK-8 detection showed that CD133(-) subgroup of MKN45 GC cell line was more sensitive than CD133(+) cells to 5-FU (P<0.05). Hoechst 33258 staining showed that there were more apoptotic cells in CD133(-) subgroup as compared to other two subgroups, and the least apoptotic cells were observed in CD133(+) subgroup of MKN45 GC cell line (P<0.05). CD133 sirna was transfected into MKN45 GC cell line and could down-regulate the expressions of CD133, P-gp, Bcl-2 and p-Akt, while the expression of Bax increased (all P<0.05).

CONCLUSIONSCD133 may contribute to the resistance of GC cells to chemotherapy drug through P-gp, Bcl-2 and Bax. PI3K/Akt signal pathway may be involved in this process.

AC133 Antigen ; ATP-Binding Cassette, Sub-Family B, Member 1 ; Antigens, CD ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Fluorouracil ; Glycoproteins ; metabolism ; Humans ; Peptides ; metabolism ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; RNA, Small Interfering ; Stomach Neoplasms ; drug therapy ; metabolism ; pathology ; bcl-2-Associated X Protein